Methods for the Production and Use of Myceliated High Protein Food Compositions

ABSTRACT

Disclosed is a method to prepare a myceliated high-protein food product, which includes culturing a fungi an aqueous media which has a high level of plant protein, for example at least 20 g protein per 100 g dry weight with excipients, on a dry weight basis. The plant protein can include pea, rice and/or chickpea. The fungi can include comprises  Lentinula  spp.,  Agaricus  spp.,  Pleurotus  spp.,  Boletus  spp., or  Laetiporus  spp. After culturing, the material is harvested by obtaining the myceliated high-protein food product via drying or concentrating. The resultant myceliated high-protein food product may have its taste, flavor, or aroma modulated, such as by increasing desirable flavors or tastes such as meaty, savory, umami, popcorn and/or by decreasing undesirable flavors such as bitterness, astringency or beaniness. Deflavoring and/or deodorizing as compared to non-myceliated control materials can also be observed. Also disclosed are myceliated high-protein food products.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of pending U.S. Ser. No. 15/488,183,filed Apr. 14, 2017, entitled “Methods for the Production and use ofMyceliated High Protein Food Compositions,” which is a regular utilityapplication filed from U.S. Provisional Application No. 62/322,726,filed Apr. 14, 2016, now expired, entitled “Methods for the productionand use of Myceliated High Protein Food Compositions”, the disclosure ofeach of which is hereby incorporated by reference herein in itsentirety.

BACKGROUND OF INVENTION

There is a growing need for efficient, high quality and low costhigh-protein food sources with acceptable taste, flavor and/or aromaprofiles. However, it has proven difficult to achieve such products,particularly with low cost vegetarian protein sources.

Previous work discloses culturing of fungi using low amounts of proteinin the culture media. U.S. Pat. No. 2,693,665 discusses the submergedculturing of Agaricus campestris grown in citrus juice, pear juice,asparagus juice, “organic material”, a carbohydrate, a nitrogen source,and any combination of these materials optionally supplemented with ureaand/or various ammonium salts.

U.S. Pat. No. 2,761,246 teaches a method for the production of submergedMorchella esculenta, and more broadly Helvellaceae mycelium for thepurposes of creating a human foodstuff. The publication discusses theuse of various molasses solutions as media supplemented with ammoniumsalts and the inclusion of calcium carbonate or calcium sulfate asnucleation sites for hyphal spheres to increase biomass yield 30 fold.In general, the patent teaches the art of growing submerged mycelium ona carbohydrate source “such as many of the monosaccharides, or some ofthe disaccharides or their hydrolysates” and a nitrogen source “such asammonium salts or amino acids or any kind of protein hydrolysate”. Theculture propagation motif includes three separate cultures and anintermittent filtering step.

U.S. Pat. No. 2,928,210 discloses a method to produce mushroom myceliumfrom sulfite liquor waste supplemented with organic and inorganic salts,presenting the idea as an efficient way to prevent sulfite liquorpollution. Culture propagation does require that the mycelium be washedto remove residual liquor, taught as a necessary step to make theproduct human food grade. This introduces the disadvantage of washingaway exocellular solids that would otherwise greatly contribute toyield. This also introduces a new waste stream that will presents thesame problems the publication is trying to solve.

U.S. Pat. No. 3,086,320 discloses a method to improve the flavor ofsubmerged mycelium of M. ‘esculema’, Helvella gigas, Coprinus comatus,and A. campestris by growing the strains in a media comprising milk. Thepatent claims the major issue of edible mycelium is that “the mycelium,while similar in flavor to the natural sporophore, falls short inmatching it in intensity and kind.” The patent teaches the use of 1 to50% (v/v) natural skim milk to media, or 0.33 to 16.66% condensednatural skim milk with nonfat dry milk solids in an amount of about 0.03to 1.66% (w/w) to the condensed natural skim milk if the condensed milkis being substituted for the non-condensed. If using natural skim milk,milk protein hydrolysate can be used in an amount of about 5% (w/w). Thepatent recommends using skim milk in an amount between 5-10% (v/v) tomedia. Mycelium flavor is said to improve with higher concentrations ofmilk.

U.S. Pat. No. 4,071,973 discloses culturing conditions forBasidiomycetes. The patent teaches to inoculate media with “a body of afungus” and supply “inorganic nutrient salts for nitrogen, phosphate andpotassium,” mixed with sucrose at 50-70 g/L and supplemented with finepowder of “crushed sugarcane, sugarcane bagasse, pine tree-tissue andwheat bran” at 0.2-15 g/L. Oxygen is controlled at 30-90% (v/v) to themedia and the vessel is pressurized at 0.12-0.5 MPa (17.4 to 72.5 psi)with oxygen supplied at 0.1 to 1.0 L/minute. Salts used include ammoniumnitrate, sodium phosphate, magnesium sulfate heptahydrate, iron (II)sulfate heptahydrate and dipotassium hydrogen phosphate. Air pressurecycles are controlled with a pressure regulator. The patent states thatcell growth enhancement through elevated oxygen levels is unexpected.

There is therefore a need for efficient, high quality and low costhigh-protein food sources with acceptable taste, flavor and/or aromaprofiles, and for a process that enables the myceliation of highlyproteinaceous media, specifically media that are greater than 50%protein on a dry weight basis.

SUMMARY OF THE INVENTION

The present inventors have found that culturing a fungus in a highprotein media provides an economically viable product, and also foundthat such treatment can also alter the taste, flavor or aroma of highprotein food compositions in unexpected ways. The process additionallyenables the production of protein concentrates, isolates and highprotein foodstuffs that have been imbued with mycelial material, therebyaltering aspects of the media used in the production of productsaccording to the methods of the present invention. The present inventionalso presents the ability to stack protein sources to optimize aminoacid profiles of products made according to the methods of theinvention.

Thus, the present invention includes methods to prepare a myceliatedhigh-protein food product by culturing a fungus in an aqueous mediawhich includes a high-protein material, with amounts of protein of atleast 20 g protein per 100 g total dry weight with excipients, resultingin a myceliated high-protein food product, whereby the flavor or tasteof the myceliated high-protein food product is modulated compared to thehigh-protein material.

Appropriate fungi to use in the methods of the present inventioninclude, for example, Lentinula spp., such as L. edodes, Agaricus spp.,such as A. blazei, A. bisporus, A. campestris, A. subrufescens, A.brasiliensis, or A. silvaticus; Pleurotus spp., Boletus spp., orLaetiporus spp. In one embodiment, the fungi for the invention includefungi from optionally, liquid culture of species generally known asoyster, porcini, ‘chicken of the woods’ and shiitake mushrooms. Theseinclude Pleurotus (oyster) species such as Pleurotus ostreatus,Pleurotus salmoneostramineus (Pleurotus djamor), Pleurotus eryngii, orPleurotus citrinopileatus; Boletus (porcini) species such as Boletusedulis; Laetiporus (chicken of the woods) species such as Laetiporussulfureus, and many others such as L. budonii, L. miniatus, L.flos-musae, L. discolor; and Lentinula (shiitake) species such as L.edodes. Also included are Lepista nuda, Hericium erinaceus, Agaricusblazeii, and combinations thereof.

The amounts of protein in the aqueous media can be between 10 g/Lprotein and 500 g/L protein. The aqueous media may include ahigh-protein material, which is a protein concentrate or a proteinisolate from a vegetarian or non-vegetarian source. The vegetariansource may include pea, rice, soy, cyanobacteria, grain, hemp, chia,chickpea, potato protein, algal protein and nettle protein orcombinations of these. In embodiments, the vegetarian source is pea,rice, chickpea or a combination thereof. In embodiments, the vegetariansource is pea, chickpea or a combination thereof. In embodiments, thevegetarian source is rice, chickpea, or a combination thereof.

The produced myceliated high-protein food product may be pasteurized,sterilized, dried, powderized. The produced myceliated high-protein foodproduct may have its flavors, tastes, and/or aromas enhanced, such as byincreasing meaty flavors, enhancing umami taste, enhancing savoryflavors, enhancing popcorn flavors, or enhancing mushroom flavors in themyceliated high-protein food product; or, the produced high-protein foodproduct may have its flavors, tastes and/or aromas decreased, resultingin milder aromas or tastes, or reduced bitter, astringent, beanyflavors, tastes, or aromas.

In embodiments, the aromas reduced include a reduced pea aroma, areduced rice aroma, a reduced beany aroma, a reduced mushroom aroma, areduced overripe vegetable aroma, or decreased cardboard-type aroma. Insome embodiments, the myceliated high protein food product has increasedmushroom aroma. In embodiments, a myceliated high protein food productthat includes pea protein will have reduced pea aroma; or a myceliatedhigh protein food product that includes rice protein will have reducedrice aroma. In embodiments, a myceliated high protein food product willhave an increased mushroom aroma.

In embodiments, the flavors reduced include reduced pea flavor, reducedbeany flavor, reduced rice flavor. In embodiments, the flavors increasedinclude increased sour flavors, increased umami flavors, increasedmushroom flavors.

The present invention also includes a myceliated high-protein foodproduct made by, for example, the processes of the invention. Themyceliated high-protein food product may be at least 20% protein, may beproduced from a vegetarian source such as pea or rice, and may haveenhanced desirable flavors and/or decreased undesirable

Without wishing to be bound by any particular theory, there may bediscussion herein of beliefs or understandings of underlying principlesrelating to the devices and methods disclosed herein. It is recognizedthat regardless of the ultimate correctness of any mechanisticexplanation or hypothesis, an embodiment of the invention cannonetheless be operative and useful.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. A 100× photomicrograph of a media containing rice and peaprotein prior to inoculation by mycelial culture.

FIG. 2. A 100× photomicrograph of a mycelial culture at Day 4 grown inmedia containing rice and pea protein.

FIG. 3. A 100× photomicrograph of a mycelial culture at Day 10 grown inmedia containing rice and pea protein.

FIG. 4. A 100× photomicrograph of a mycelial growth of the bioreactor inMedium 1 microscopic examination, 24 hours harvest.

FIG. 5. A 100× photomicrograph of a mycelial growth of the bioreactor inMedium 2 microscopic examination, 44 hours harvest.

FIG. 6. A 100× photomicrograph of a mycelial growth of the bioreactor inMedium 3, Microscopic examination, 30 hours harvest.

DETAILED DESCRIPTION OF THE INVENTION

In general, the terms and phrases used herein have their art-recognizedmeaning, which can be found by reference to standard texts, journalreferences and contexts known to those skilled in the art. The followingdefinitions are provided to clarify their specific use in the context ofthe invention.

In one embodiment, the present invention includes a method to prepare amyceliated high-protein food product. The method may optionally includethe steps of providing an aqueous media comprising a high-proteinmaterial. The aqueous media may comprise, consist of, or consistessentially of at least 20 g protein per 100 g total excipients, on adry weight basis. The media may also comprise, consist of or consistessentially of optional additional excipients as identified hereinbelow.The aqueous media may be inoculated with a fungal culture. Theinoculated media may then be cultured to produce a myceliatedhigh-protein food product, and the myceliated high-protein food producttaste, flavor, and/or aroma may be modulated compared to thehigh-protein material in the absence of the culturing step.

The aqueous media may comprise, consist of, or consist essentially of ahigh-protein material. The high-protein material to include in theaqueous media can be obtained from a number of sources, includingvegetarian sources (e.g., plant sources) as well as non-vegetariansources, and can include a protein concentrate and/or isolate.Vegetarian sources include meal, protein concentrates and isolatesprepared from a vegetarian source such as pea, rice, chickpea, soy,cyanobacteria, hemp, chia and other sources, or a combination thereof.For example, cyanobacteria containing more than 50% protein can also beused a source of high-protein material. Typically, a protein concentrateis made by removing the oil and most of the soluble sugars from a meal,such as soybean meal. Such a protein concentrate may still contain asignificant portion of non-protein material, such as fiber. Typically,protein concentrations in such products are between 55-90%. The processfor production of a protein isolate typically removes most of thenon-protein material such as fiber and may contain up to about 90-99%protein. A typical protein isolate is typically subsequently dried andis available in a powdered form and may alternatively be called “proteinpowder.”

Non-vegetarian sources for the high-protein material may also be used inthe present invention. Such non-vegetarian sources include whey, casein,egg, meat (beef, chicken, pork sources, for example), isolates,concentrates, broths, or powders. However, in some embodimentsvegetarian sources have certain advantages over non-vegetarian sources.For example, whey or casein protein isolates generally contain someamount of lactose and which can cause difficulties for those who arelactose-intolerant. Egg protein isolates may cause problems to those whoare allergic to eggs and are is also quite expensive. Certain vegetablesources have disadvantages as well, while soy protein isolates have goodProtein Digestibility Corrected Amino Acid Scores (PDCAAS) anddigestible indispensable amino acid scores (DIAAS) scores, and isinexpensive, soy may be allergenic and has some consumer resistance dueto concerns over phytoestrogens and taste. Rice protein is highlydigestible, but is deficient in some amino acids such as lysine. Riceprotein is therefore not a complete protein and further many peopleperceive rice protein to have an off-putting taste and aroma. Peaprotein is generally considered to contain all essential amino acids, isnot balanced and thus is not complete and many people perceive peaprotein to have an off-putting aroma. Hemp protein is a complete proteinwith decent taste and aroma, but is expensive.

In one embodiment, mixtures of any of the high-protein materialsdisclosed can be used to provide, for example, favorable qualities, suchas a more complete (in terms of amino acid composition) high-proteinmaterial. In one embodiment, high-protein materials such as pea proteinand rice protein can be combined. In one embodiment, the ratio of amixture can be from 1:10 to 10:1 pea protein:rice protein (on a drybasis). In one embodiment, the ratios can optionally be 5:1 to 1:5, 2:1to 1:2, or in one embodiment, 1:1.

The high-protein material itself can be about 20% protein, 30% protein,40% protein, 45% protein, 50% protein, 55% protein, 60% protein, 65%protein, 70% protein, 75% protein, 80% protein, 85% protein, 90%protein, 95% protein, or 98% protein, or at least about 20% protein, atleast about 30% protein, at least about 40% protein, at least about 45%protein, at least about 50% protein, at least about 55% protein, atleast about 60% protein, at least about 65% protein, at least about 70%protein, at least about 75% protein, at least about 80% protein, atleast about 85% protein, at least about 90% protein, at least about 95%protein, or at least about 98% protein.

This invention discloses the use of concentrated media, which provides,for example, an economically viable economic process for production ofan acceptably tasting and/or flavored high-protein food product. In oneembodiment of the invention the total media concentration is up to 150g/L but can also be performed at lower levels, such as 5 g/L. Higherconcentrations in media result in a thicker and/or more viscous media,and therefore are optionally processed by methods known in the art toavoid engineering issues during culturing or fermentation. To maximizeeconomic benefits, a greater amount of high-protein material per L mediais used. The amount is used is chosen to maximize the amount ofhigh-protein material that is cultured, while minimizing technicaldifficulties in processing that may arise during culturing such asviscosity, foaming and the like. The amount to use can be determined byone of skill in the art, and will vary depending on the method offermentation

The amount of total protein in the aqueous media may comprise, consistof, or consist essentially of at least 20 g, 25 g, 30 g, 35 g, 40 g, 45g, 50 g, 55 g, 60 g, 65 g, 70 g, 75 g, 80 g, 85 g, 90 g, 95 g, or 100 g,or more, of protein per 100 g total dry weight with excipients, or pertotal all components on a dry weight basis. Alternatively, the amount ofprotein comprise, consist of, or consist essentially of between 20 g to90 g, between 30 g and 80 g, between 40 g and 70 g, between 50 g and 60g, of protein per 100 g dry weight with excipients.

In some embodiments, the total protein in aqueous media is about 45 g toabout 100 g, or about 80-100 g of protein per 100 g dry weight withexcipients.

In another embodiment, the aqueous media comprises between about 1 g/Land 200 g/L, between about 5 g/L and 180 g/L, between about 20 g/L and150 g/L, between about 25 g/L and about 140 g/L, between about 30 g/Land about 130 g/L, between about 35 g/L and about 120 g/L, between about40 g/L and about 110 g/L, between about 45 g/L and about 105 g/L,between about 50 g/L and about 100 g/L, between about 55 g/L and about90 g/L, or about 75 g/L protein; or between about 50 g/L-150 g/L, orabout 75 g/L and about 120 g/L, or about 85 g/L and about 100 g/L.Alternatively, the aqueous media comprises at least about 10 g/L, atleast about 15 g/L, at least about 20 g/L, at least about 25 g/L, atleast about 30 g/L, at least about 35 g/L, at least about 40 g/L or atleast about 45 g/L protein. In fermenters, in some embodiments theamount to use includes between about 1 g/L and 150 g/L, between about 10g/L and 140 g/L, between about 20 g/L and 130 g/L, between about 30 g/Land about 120 g/L, between about 40 g/L and about 110 g/L, between about50 g/L and about 100 g/L, between about 60 g/L and about 90 g/L, betweenabout 70 g/L and about 80 g/L, or at least about 20 g/L, at least about30 g/L, at least about 40 g/L, at least about 50 g/L, at least about 60g/L, at least about 70 g/L, at least about 80 g/L, at least about 90g/L, at least about 100 g/L, at least about 110 g/L, at least about 120g/L, at least about 130 g/L or at least about 140 g/L.

In some embodiments, the aqueous media comprises between about 50 g/Land about 100 g/L, or about 80 g/L, about 85 g/L, about 90 g/L, about 95g/L about 100 g/L, about 110 g/L, about 120 g/L, about 130 g/L, about140 g/L, or about 150 g/L.

It can be appreciated that in calculating such percentages, thepercentage of protein in the high-protein material must accounted for.For example, if the amount of high-protein material is 10 g, and thehigh-protein material is 80% protein, then the protein source includes 8g protein and 2 non-protein material. When added to 10 g of excipientsto create 20 total grams dry weight with excipients, then the total is 8g protein per 20 g total excipients, or 40% protein, or 40 g protein per100 g total protein plus excipients. If a protein-containing excipientsuch as yeast extract or peptone is added to the media, the amount ofprotein per g total weight plus excipients will be slightly higher,taking into account the percentage of protein and the amount added ofthe protein-containing excipient, and performing the calculation asdiscussed herein, as is known in the art.

In some embodiments, the high-protein material, after preparing theaqueous media of the invention, is not completely dissolved in theaqueous media. Instead, the high-protein material may be partiallydissolved, and/or partially suspended, and/or partially colloidal.However, even in the absence of complete dissolution of the high-proteinmaterial, positive changes may be affected during culturing of thehigh-protein material. In one embodiment, the high-protein material inthe aqueous media is kept as homogenous as possible during culturing,such as by ensuring agitation and/or shaking.

In one embodiment, the aqueous media further comprises, consists of, orconsists essentially of materials other than the high-protein material,e.g., excipients as defined herein and/or in particular embodiments.Excipients can comprise any other components known in the art topotentiate and/or support fungal growth, and can include, for example,nutrients, such as proteins/peptides, amino acids as known in the artand extracts, such as malt extracts, meat broths, peptones, yeastextracts and the like; energy sources known in the art, such ascarbohydrates; essential metals and minerals as known in the art, whichincludes, for example, calcium, magnesium, iron, trace metals,phosphates, sulphates; buffering agents as known in the art, such asphosphates, acetates, and optionally pH indicators (phenol red, forexample). Excipients may include carbohydrates and/or sources ofcarbohydrates added to media at 5-10 g/L. It is usual to add pHindicators to such formulations.

Excipients may also include peptones/proteins/peptides, as is known inthe art. These are usually added as a mixture of protein hydrolysate(peptone) and meat infusion, however, as used in the art, theseingredients are typically included at levels that result in much lowerlevels of protein in the media than is disclosed herein. Many mediahave, for example, between 1% and 5% peptone content, and between 0.1and 5% yeast extract and the like.

In one embodiment, excipients include for example, yeast extract, maltextract, maltodextrin, peptones, and salts such as diammonium phosphateand magnesium sulfate, as well as other defined and undefined componentssuch as potato or carrot powder. In some embodiments, organic (asdetermined according to the specification put forth by the NationalOrganic Program as penned by the USDA) forms of these components may beused.

In one embodiment, excipients comprise, consist of, or consistessentially of dry carrot powder, dry malt extract, diammoniumphosphate, magnesium sulfate, and citric acid. In one embodiment,excipients comprise, consist of, or consist essentially of dry carrotpowder between 0.1-10 g/L, dry malt extract between 0.1 and 20 g/L,diammonium phosphate between 0.1 and 10 g/L, and magnesium sulfatebetween 0.1 and 10 g/L. Excipients may also optionally comprise, consistof, or consist essentially of citric acid and an anti-foam component.The anti-foam component can any anti-foam component known in the art,such as a food-grade silicone anti-foam emulsion or an organic polymeranti-foam (such as a polypropylene-based polyether composition).

In another embodiment, the medium comprises, consists of or consistsessentially of the high protein material as defined herein and ananti-foam component, without any other excipients present.

The method may also comprise the optional step of sterilizing theaqueous media prior to inoculation by methods known in the art,including steam sterilization and all other known methods to allow forsterile procedure to be followed throughout the inoculation andculturing steps to enable culturing and myceliation by pure fungalstrains. Alternatively, the components of the media may be separatelysterilized and the media may be prepared according to sterile procedure.

The method also includes inoculating the media with a fungal culture.The fungal culture may be prepared by culturing by any methods known inthe art. In one embodiment, the methods to culture may be found in,e.g., PCT/US14/29989, filed Mar. 15, 2014, PCT/US14/29998, filed Mar.15, 2014, all of which are incorporated by reference herein in theirentireties.

The fungal cultures, prior to the inoculation step, may be propagatedand maintained as is known in the art. In one embodiment, the fungidiscussed herein can be kept on 2-3% (v/v) mango puree with 3-4% agar(m/v). Such media is typically prepared in 21.6 L handled glass jarsbeing filled with 1.4-1.5 L media. Such a container pours for 50-60 90mm Petri plates. The media is first sterilized by methods known in theart, typically with an autoclave. Conventional B. stearothermophilus andthermocouple methods are used to verify sterilization parameters. Somestrains, such as L. sulfureus, grow better when supplemented with 1%yellow cornmeal. Agar media can also be composed of high-proteinmaterial to sensitize the strain to the final culture. This techniquemay also be involved in strain selection of the organisms discussedherein. Agar media should be poured when it has cooled to the pointwhere it can be touched by hand (˜40-50° C.).

In one embodiment, maintaining and propagating fungi for use forinoculating the high-protein material as disclosed in the presentinvention may be carried out as follows. For example, a propagationscheme that can be used to continuously produce material according tothe methods is discussed herein. Once inoculated with master culture andsubsequently colonized, Petri plate cultures can be used at any point topropagate mycelium into prepared liquid media. As such, plates can bepropagated at any point during log phase or stationary phase but areencouraged to be used within three months and in another embodimentwithin 2 years, though if properly handled by those skilled in the artcan generally be stored for as long as 10 years at 4° C. and up to 6years at room temperature.

In some embodiments, liquid cultures used to maintain and propagatefungi for use for inoculating the high-protein material as disclosed inthe present invention include undefined agricultural media with optionalsupplements as a motif to prepare culture for the purposes ofinoculating solid-state material or larger volumes of liquid. In someembodiments, liquid media preparations are made as disclosed herein.Liquid media can be also sterilized and cooled similarly to agar media.Like agar media it can theoretically be inoculated with any fungalculture so long as it is deliberate and not contaminated with anyundesirable organisms (fungi inoculated with diazotrophs may bedesirable for the method of the present invention). As such, liquidmedia are typically inoculated with agar, liquid and other forms ofculture. Bioreactors provide the ability to monitor and controlaeration, foam, temperature, and pH and other parameters of the cultureand as such enables shorter myceliation times and the opportunity tomake more concentrated media.

In one embodiment, the fungi for use for inoculating the high-proteinmaterial as disclosed in the present invention may be prepared as asubmerged liquid culture and agitated on a shaker table, or may beprepared in a shaker flask, by methods known in the art and according tomedia recipes disclosed in the present invention. The fungal componentfor use in inoculating the aqueous media of the present invention may bemade by any method known in the art. In one embodiment, the fungalcomponent may be prepared from a glycerol stock, by a simple propagationmotif of Petri plate culture to 0.5-−4 L Erlenmeyer shake flask to 50%glycerol stock. Petri plates can comprise agar in 10-35 g/L in additionto various media components. Conducted in sterile operation, chosenPetri plates growing anywhere from 1-˜3,652 days can be propagated into0.5-4 L Erlenmeyer flasks (or 250 to 1,000 mL Wheaton jars, or anysuitable glassware) for incubation on a shaker table or stationaryincubation. The smaller the container, the faster the shaker should be.In one embodiment, the shaking is anywhere from 40-160 RPM depending oncontainer size and, with about a 1″ swing radius.

The culturing step of the present invention may be performed by methods(such as sterile procedure) known in the art and disclosed herein andmay be carried out in a fermenter, shake flask, bioreactor, or othermethods. In a shake flask, in one embodiment, the agitation rate is 50to 240 RPM, or 85 to 95 RPM, and incubated for 1 to 90 days. In anotherembodiment the incubation temperature is 70-90° F. In another embodimentthe incubation temperature is 87-89° F. Liquid-state fermentationagitation and swirling techniques as known in the art are also employedwhich include mechanical shearing using magnetic stir bars, stainlesssteel impellers, injection of sterile high-pressure air, the use ofshaker tables and other methods such as lighting regimen, batch feedingor chemostatic culturing, as known in the art.

In one embodiment, culturing step is carried out in a bioreactor whichis ideally constructed with a torispherical dome, cylindrical body, andspherical cap base, jacketed about the body, equipped with a magneticdrive mixer, and ports to provide access for equipment comprising DO,pH, temperature, level and conductivity meters as is known in the art.Any vessel capable of executing the methods of the present invention maybe used. In another embodiment the set-up provides 0.1-5.0 ACH. Otherengineering schemes known to those skilled in the art may also be used.

The reactor can be outfitted to be filled with water. The water supplysystem is ideally water for injection (WFI) system, with a sterilizableline between the still and the reactor, though RO or any potable watersource may be used so long as the water is sterile. In one embodimentthe entire media is sterilized in situ while in another embodimentconcentrated media is sterilized and diluted into a vessel filled waterthat was filter and/or heat sterilized, or sufficiently treated so thatit doesn't encourage contamination over the colonizing fungus. Inanother embodiment, high temperature high pressure sterilizations arefast enough to be not detrimental to the media. In one embodiment theentire media is sterilized in continuous mode by applying hightemperature between 130° and 150° C. for a residence time of 1 to 15minutes. Once prepared with a working volume of sterile media, the tankcan be mildly agitated and inoculated. Either as a concentrate or wholemedia volume in situ, the media can be heat sterilized by steamingeither the jacket, chamber or both while the media is optionallyagitated. The medium may optionally be pasteurized instead.

In one embodiment, the reactor is used at a large volume, such as in500,000-200,000 L working volume bioreactors. When preparing material atsuch volumes the culture must pass through a successive series of largerbioreactors, any bioreactor being inoculated at 0.5-15% of the workingvolume according to the parameters of the seed train. A typical processwould pass a culture from master culture, to Petri plates, to flasks, toseed bioreactors to the final main bioreactor when scaling the method ofthe present invention. To reach large volumes, 3-4 seeds may be used.The media of the seed can be the same or different as the media in themain. In one embodiment, the fungal culture for the seed is a proteinconcentration as defined herein, to assist the fungal culture inadapting to high-protein media in preparation for the main fermentation.Such techniques are discussed somewhat in the examples below. In oneembodiment, foaming is minimized by use of anti-foam on the order of 0.5to 2.5 g/L of media, such as those known in the art, including insolubleoils, polydimethylsiloxanes and other silicones, certain alcohols,stearates and glycols. In one embodiment, lowering pH assists in culturegrowth, for example, for L. edodes pH may be adjusted by use of citricacid or by any other compound known in the art, but care must be takento avoid a sour taste for the myceliated high-protein product. The pHmay be adjusted to between about 4.5 and 5.5, for example, to assist ingrowth.

In one embodiment, during the myceliation step, for example, wherein themedia comprises at least 50% (w/w) protein on a dry weight basis, and/orwherein the media comprises at least 50 g/L protein, the pH does notchange during processing. “pH does not change during processing” isunderstood to mean that the pH does not change in any significant way,taking into account variations in measured pH which are due toinstrument variations and/or error. For example, the pH will stay withinabout plus or minus 0.3 pH units, plus or minus 0.25 pH units, plus orminus 0.2 pH units, plus or minus 0.15 pH units, or plus or minus 0.1 pHunits of a starting pH of the culture during the myceliation, e.g.processing step. Minor changes in pH are also contemplated duringprocessing, particularly in media which do not contain an exogenousbuffer such as diammonium phosphate. A minor change in pH can be definedas a pH change of plus or minus 0.5 pH units or less, plus or minus 0.4pH units or less, plus or minus 0.3 pH units or less, plus or minus 0.25pH units or less, plus or minus 0.2 pH units or less, plus or minus 0.15pH units or less, or plus or minus 0.1 pH units or less of a startingpH.

FIGS. 1-3 show an exemplification of the preparation of L. edodes as thefungal component for use for inoculating an aqueous media to prepare themyceliated high-protein food product. In this embodiment, a 1:1 mixtureof pea protein and rice protein at 40% protein (8 g per 20 g total plusexcipients) media was prepared, and FIGS. 1-3 show the results ofconducting microscope checks over time (0, 4 and 10 days). The increasein biomass concentration was correlated with a drop in pH. After shakingfor 1 to 10 days, an aliquot (e.g. 10 to 500 mL) of the shake flask maybe transferred in using sterile procedure into a sterile, preparedsealed container (such as a customized stainless steel can orappropriate conical tube), which can then adjusted with about 5-60%,sterile, room temperature (v/v) glycerol. The glycerol stocks can may besealed with a water tight seal and can be held stored at −20° C. forstorage. The freezer is ideally a constant temperature freezer. Glycerolstocks stored at 4° C. may also be used. Agar cultures can be used asinoculant for the methods of the present invention, as can any culturepropagation technique known in the art.

It was found that not all fungi are capable of growing in media asdescribed herein. Fungi useful for the present invention are from thehigher order Basidio- and Ascomycetes. In some embodiments, fungieffective for use in the present invention include, but are not limitedto, Lentinula spp., such as L. edodes, Agaricus spp., such as A. blazei,A. bisporus, A. campestris, A. subrufescens, A. brasiliensis, or A.silvaticus; Pleurotus spp., Boletus spp., or Laetiporus spp. In oneembodiment, the fungi for the invention include fungi from optionally,liquid culture of species generally known as oyster, porcini, ‘chickenof the woods’ and shiitake mushrooms. These include Pleurotus (oyster)species such as Pleurotus ostreatus, Pleurotus salmoneostramineus(Pleurotus djamor), Pleurotus eryngii, or Pleurotus citrinopileatus;Boletus (porcini) species such as Boletus edulis; Laetiporus (chicken ofthe woods) species such as Laetiporus sulfureus, and many others such asL. budonii, L. miniatus, L. flos-musae, L. discolor; and Lentinula(shiitake) species such as L. edodes. Also included are Lepista nuda,Hericium erinaceus, Agaricus blazeii, and combinations thereof. In oneembodiment, the fungi is Lentinula edodes. Fungi may be obtainedcommercially, for example, from the Penn State Mushroom CultureCollection. Strains are typically received as “master culture” PDYslants in 50 mL test tubes and are stored at all, but for A. blazeii,stored at 4° C. until plated. For plating, small pieces of culture aretypically transferred into sterile shake flasks (e.g. 250 mL) so as notto contaminate the flask filled with a sterilized media (liquid mediarecipes are discussed below). Inoculated flasks shake for approximatelyten hours and aliquots of said flasks are then plated onto preparedPetri plates of a sterile agar media. One flask can be used to preparedozens to potentially hundreds of Petri plate cultures. There are othermethods of propagating master culture though the inventors find thesemethods as disclosed to be simple and efficient.

Determining when to end the culturing step and to harvest the myceliatedhigh-protein food product, which according to the present invention, toresult in a myceliated high-protein food product with acceptable taste,flavor and/or aroma profiles, can be determined in accordance with anyone of a number of factors as defined herein, such as, for example,visual inspection of mycelia, microscope inspection of mycelia, pHchanges, changes in dissolved oxygen content, changes in proteincontent, amount of biomass produced, and/or assessment of taste profile,flavor profile, or aroma profile. In one embodiment, harvest can bedetermined by tracking protein content during culturing and harvestbefore significant catabolism of protein occurs. The present inventorsfound that protein catabolism can initiate in bioreactors at 30-50 hoursof culturing under conditions defined herein. In another embodiment,production of a certain amount of biomass may be the criteria used forharvest. For example, biomass may be measured by filtering, such througha filter of 10-1000 μm, and has a protein concentration between 0.1 and25 g/L; or in one embodiment, about 0.2-0.4 g/L. In one embodiment,harvest can occur when the dissolved oxygen reaches about 10% to about90% dissolved oxygen, or less than about 80% of the starting dissolvedoxygen. Additionally, mycelial products may be measured as a proxy formycelial growth, such as, total reducing sugars (usually a 40-95%reduction), β-glucan and/or chitin formation; harvest is indicated at10²-10⁴ ppm. Other indicators include small molecule metaboliteproduction depending on the strain (e.g. eritadenine on the order of0.1-20 ppm for L. edodes or erinacine on the order of 0.1-1,000 ppm forH. erinaceus) or nitrogen utilization (monitoring through the use of anynitrogenous salts or protein, cultures may be stopped just as proteinstarts to get utilized or may continue to culture to enhance thepresence of mycelial metabolites). In one embodiment, the total proteinyield in the myceliated high-protein food product after the culturingstep is about 75% to about 95%.

Harvest includes obtaining the myceliated high-protein food productwhich is the result of the myceliation step. After harvest, cultures canbe processed according to a variety of methods. In one embodiment, themyceliated high-protein food product is pasteurized or sterilized. Inone embodiment, the myceliated high-protein food product is driedaccording to methods as known in the art. Additionally, concentrates andisolates of the material may be prepared using variety of solvents orother processing techniques known in the art. In one embodiment thematerial is pasteurized or sterilized, dried and powdered by methodsknown in the art. Drying can be done in a desiccator, vacuum dryer,conical dryer, spray dryer, fluid bed or any method known in the art.Preferably, methods are chosen that yield a dried myeliated high-proteinproduct (e.g., a powder) with the greatest digestibility andbioavailability. The dried myeliated high-protein product can beoptionally blended, pestled milled or pulverized, or other methods asknown in the art.

In many cases, the flavor, taste and/or aroma of high-protein materialsas disclosed herein, such as protein concentrates or isolates fromvegetarian or nonvegetarian sources (e.g. egg, whey, casein, beef, soy,rice, hemp, pea, chickpea, soy, cyanobacteria, and chia) may haveflavors, which are often perceived as unpleasant, having pungent aromasand bitter or astringent tastes. These undesirable flavors and tastesare associated with their source(s) and/or their processing, and theseflavors or tastes can be difficult or impossible to mask or disguisewith other flavoring agents. The present invention, as explained in moredetail below, works to modulate these tastes and/or flavors.

In one embodiment of the invention, flavors and/or tastes of themyceliated high-protein food product or products are modulated ascompared to the high-protein material (starting material). In someembodiments, both the sterilization and myceliation contribute to themodulation of the resultant myceliated high-protein food products'taste.

In one embodiment, the aromas of the resultant myceliated high-proteinfood products prepared according to the invention are reduced and/orimproved as compared to the high-protein material (starting material).In other words, undesired aromas are reduced and/or desired aromas areincreased. In another embodiment, flavors and/or tastes may be reducedand/or improved. For example, desirable flavors and/or tastes may beincreased or added to the high-protein material by the processes of theinvention, resulting in myceliated high-protein food products that haveadded mushroom, meaty, umami, popcorn, buttery, and/or other flavors ortastes to the food product. The increase in desirable flavors and/ortastes may be rated as an increase of 1 or more out of a scale of 5 (1being no taste, 5 being a very strong taste.)

Flavors and/or tastes of myceliated high-protein food products may alsobe improved by processes of the current invention. For example,deflavoring can be achieved, resulting in a milder flavor and/or withthe reduction of, for example, bitter and/or astringent tastes and/orbeany and/or weedy and/or grassy tastes. The decrease in undesirableflavors and/or tastes as disclosed herein may be rated as an decrease of1 or more out of a scale of 5 (1 being no taste, 5 being a very strongtaste.)

Culturing times and/or conditions can be adjusted to achieve the desiredaroma, flavor and/or taste outcomes. For example, cultures grown forapproximately 2-3 days can yield a deflavored product whereas culturesgrown for longer may develop various aromas that can change/intensify asthe culture grows. As compared to the control and/or high-proteinmaterial, and/or the pasteurized, dried and powdered medium notsubjected to sterilization or myceliation, the resulting myceliatedhigh-protein food product in some embodiments is less bitter and has amore mild, less beany aroma.

In one embodiment of the present invention, the myceliated high-proteinfood products made by the methods of the invention have a complete aminoacid profile (all amino acids in the required daily amount) because ofthe media from which it was made has such a profile. While amino acidand amino acid profile transformations are possible according to themethods of the present invention, many of the products made according tothe methods of the present invention conserve the amino acid profilewhile at the same time, more often altering the molecular weightdistribution of the proteome.

In one embodiment, when grown in a rice and pea protein concentratemedium the oyster fungi (Pleurotus ostreatus) can convey a strong savoryaroma that leaves after a few seconds at which point a mushroom flavoris noticeable. In one embodiment, the strains convey a savory meatyaroma and/or umami, savory or meaty flavor and/or taste. L. edodes andA. blazeii in some embodiments are effective at deflavoring with shorterculturing times, such as 1.5-8 days, depending on whether the culture isin a shake flask or bioreactor. L. edodes to particularly good for thedeflavoring of pea and rice protein concentrate mixtures.

In one embodiment of the instant invention, a gluten isolate orconcentrate can be mixed into a solution with excipients as disclosedherein in aqueous solution. In one embodiment, the gluten content of themedium is ≥10% (10-100%) on a dry weight basis and sterilized by methodsknown in the art for inoculation by any method known in the art with anyfungi disclosed herein, for example, with L. sulfureus. It has beenfound that L. sulfureus produces large amounts of guanosinemonophosphate (GMP) (20-40 g/L) and gluten hydrolysate, and it istheorized that the process of culturing will result in loweringmeasurable gluten content, such as below 20 ppm gluten on a dry weightbasis according to ELISA assay. Without being bound by theory, it isbelieved that the cultured material, by action of production of GMP andgluten hydrolysate, act synergistically to produce umami flavor. Withoutbeing bound by theory, it is believed that the combination of GMP andgluten hydrolysate amplifies the umami intensity in some kind ofmultiplicative as opposed to additive manner. The culture can beprocessed by any of methods disclosed in the invention and as are knownin the art to produce a product of potent umami taste. Gluten may beobtained from any source known in the art, such as corn, wheat and thelike, and may be used as a concentrate or isolate from a source.

The present invention also includes a myceliated food product made byany of the methods of as disclosed herein.

The present invention also comprises a myceliated high-protein foodproduct as defined herein. The myceliated high-protein food product cancomprise, consist of, or consist essentially of at least 20%, at least25%, at least 30%, at least 35%, at least 40%, at least 45%, at least50%, at least 55%, at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, or at least 95%, protein.

“Myceliated” as used herein, means a high-protein material as definedherein having been cultured with live fungi as defined herein andachieved at least a 5%, at least a 10%, at least a 20%, at least a 30%,at least a 40%, at least a 50%, at least a 60%, at least a 70%, at leasta 80%, at least a 90%, at least a 100%, at least a 120%, at least a140%, at least a 160%, at least a 180%, at least a 200%, at least a250%, at least a 300%, at least a 400%, at least a 500% increase inbiomass or more, to result in a myceliated high-protein food product.

In some embodiments, the high-protein material is a protein concentrateor a protein isolate, which may be obtained from vegetarian ornonvegetarian source as defined herein, including pea, rice, soy, orcombinations thereof. In some embodiments, the myceliated high-proteinfood product can be myceliated by a fungal culture as defined herein. Insome embodiments, the myceliated high-protein food product can haveenhanced meaty, savory, umami, popcorn, and/or mushroom flavors, aromasand/or tastes as compared to the high-protein material. In otherembodiments, the myceliated high-protein food product has decreasedflavors, tastes and/or aromas (deflavoring) leading to a milder and/oran improved flavor, taste or aroma. In one embodiment reducedbitterness, astringency and/or beany, grassy or weedy tastes areobserved.

EXAMPLES Example 1

Eighteen (18) 1 L baffled DeLong Erlenmeyer flasks were filled with0.400 L of a medium consisting of 25 g/L organic pea protein concentrate(labeled as 80% protein), 25 g/L organic rice protein concentrate(labeled as 80% protein), 4 g/L organic dry malt extract, 2 g/Ldiammonium phosphate, 1 g/L organic carrot powder and 0.4 g/L magnesiumsulfate heptahydrate in RO water. The flasks were covered with astainless steel cap and sterilized in an autoclave on a liquid cyclethat held the flasks at 120-121° C. for 90 minutes. The flasks werecarefully transferred to a clean HEPA laminar flowhood where they cooledfor 18 hours. Sixteen (16) flasks were subsequently inoculated with 2cm² pieces of mature Petri plate cultures of P. ostreatus, P. eryngii,L. nuda, H. erinaceus, L. edodes, A. blazeii, L. sulfureus and B.edulis, each strain done in duplicate from the same plate. All 18 flaskswere placed on a shaker table at 150 rpm with a swing radius of 1″ atroom temperature. The Oyster (P. ostreatus), Blewit (Lepista nuda) andLion's Mane (H. erinaceus) cultures were all deemed complete at 72 hoursby way of visible and microscopic inspection (mycelial balls wereclearly visible in the culture, and the isolation of these ballsrevealed dense hyphal networks under a light microscope). The othersamples, but for the Porcini (Boletus edulis) which did not grow well,were harvested at 7 days. All samples showed reduced pea and reducedrice aroma and flavor, as well as less “beany” type aromas/flavors. TheOysters had a specifically intense savory taste and back-end mushroomflavor. The Blewit was similar but not quite as savory. The Lion's Manesample had a distinct ‘popcorn’ aroma. The 3, 7 day old samples werenearly considered tasteless but for the Chicken of the Woods (Laetiporussulphureus) sample product which had a nice meaty aroma and had no peaor rice aroma/flavor. The control sample smelled and tasted like acombination of pea and rice protein and was not considered desirable.The final protein content of every the resulting cultures was between50-60% and the yields were between 80-90% after desiccation andpestling.

Example 2

Three (3) 4 L Erlenmeyer flasks were filled with 1.5 L of a mediumconsisting of 5 g/L pea protein concentrate (labeled as 80% protein), 5g/L rice protein concentrate (labeled as 80% protein), 3 g/L maltextract and 1 g/L carrot powder. The flasks were wrapped with asterilizable biowrap which was wrapped with autoclave tape 5-6 times(the taped biowrap should be easily taken off and put back on the flaskwithout losing shape) and sterilized in an autoclave that held theflasks at 120-121° C. for 90 minutes. The flasks were carefullytransferred to a clean HEPA laminar flowhood where they cooled for 18hours. Each flask was subsequently inoculated with 2 cm² pieces of 60day old P1 Petri plate cultures of L. edodes and placed on a shakertable at 120 rpm with a 1″ swing radius at 26° C. After 7-15 days, theinventors noticed, by using a pH probe on 20 mL culture aliquots, thatthe pH of every culture had dropped nearly 2 points since inoculation.L. edodes is known to produce various organic acids on or close to theorder of g/L and the expression of these acids are likely what droppedthe pH in these cultures. A microscope check was done to ensure thepresence of mycelium and the culture was plated on LB media to ascertainthe extent of any bacterial contamination. While this culture could havebeen used as a food product with further processing (pasteurization andoptionally drying), the inventors typically use such cultures asinoculant for bioreactor cultures of media prepared as disclosedaccording to the methods of the present invention.

Example 3

A 7 L bioreactor was filled with 4.5 L of a medium consisting of 5 g/Lpea protein concentrate (labeled as 80% protein), 5 g/L rice proteinconcentrate (labeled as 80% protein), 3 g/L malt extract and 1 g/Lcarrot powder. Any open port on the bioreactor was wrapped with tinfoiland sterilized in an autoclave that held the bioreactor at 120-121° C.for 2 hours. The bioreactor was carefully transferred to a clean benchin a cleanroom, setup and cooled for 18 hours. The bioreactor wasinoculated with 280 mL of inoculant from a 12 day old flask as preparedin Example 2. The bioreactor had an air supply of 3.37 L/min (0.75 VVM)and held at 26° C. A kick-in/kick-out anti-foam system was setup and itwas estimated that ˜1.5 g/L anti-foam was added during the process. At˜3-4 days the inventors noticed that the pH of the culture had dropped˜1.5 points since inoculation, similar to what was observed in the flaskculture. A microscope check was done to ensure the presence of mycelium(mycelial pellets were visible by the naked eye) and the culture wasplated on LB media to ascertain the extent of any bacterialcontamination and none was observed. While this culture could have beenused as a food product with further processing (pasteurization andoptionally drying), the inventors typically use such cultures asinoculant for bioreactor cultures of media prepared as disclosedaccording to the methods of the present invention.

Example 4

A 250 L bioreactor was filled with 150 L of a medium consisting of 45g/L pea protein concentrate (labeled as 80% protein), 45 g/L riceprotein concentrate (labeled as 80% protein), 1 g/L carrot powder, 1.8g/L diammonium phosphate, 0.7 g/L magnesium sulfate heptahydrate, 1 g/Lanti-foam and 1.5 g/L citric acid and sterilized in place by methodsknown in the art, being held at 120-121° C. for 100 minutes. Thebioreactor was inoculated with 5 L of inoculant from two bioreactors asprepared in Example 3. The bioreactor had an air supply of 30 L/min (0.2VVM) and held at 26° C. The culture was harvested in 4 days uponsuccessful visible (mycelial pellets) and microscope checks. The pH ofthe culture did not change during processing but the DO dropped by 25%.The culture was plated on LB media to ascertain the extent of anybacterial contamination and none was observed. The culture was thenpasteurized at 82° C. for 30 minutes with a ramp up time of 30 minutesand a cool down time of 45 minutes to 17° C. The culture was finallyspray dried and tasted. The final product was noted to have a mild aromawith no perceptible taste at concentrations up to 10%. The product was˜75% protein on a dry weight basis.

Example 5

A 250 L bioreactor was filled with 150 L of a medium consisting of 45g/L pea protein concentrate (labeled as 80% protein), 45 g/L riceprotein concentrate (labeled as 80% protein), 1 g/L carrot powder, 1.8g/L diammonium phosphate, 0.7 g/L magnesium sulfate heptahydrate, 1 g/Lanti-foam and 1.5 g/L citric acid and sterilized in place by methodsknown in the art, being held at 120-121° C. for 100 minutes. Thebioreactor was inoculated with 5 L of inoculant from two bioreactors asprepared in Example 3. The bioreactor had an air supply of 30 L/min (0.2VVM) and held at 26° C. The culture was harvested in 2 days uponsuccessful visible (mycelial pellets) and microscope checks. The pH ofthe culture did not change during processing but the DO dropped by 25%.The culture was plated on LB media to ascertain the extent of anybacterial contamination and none was observed. The culture was thenpasteurized at 82° C. for 30 minutes with a ramp up time of 30 minutesand a cool down time of 90 minutes to 10° C. The culture was finallyconcentrated to 20% solids, spray dried and tasted. The final productwas noted to have a mild aroma with no perceptible taste atconcentrations up to 10%. The product was ˜75% protein on a dry weightbasis.

The amount of lactic acid in the final product (Product Batch 1 and 2are from to different fermentation runs) were as follows, as shown inTable 1:

TABLE 1 Product Batch Lactic Acid (g/L) 1 0.13 2 0.14

Example 6

Eight (8) 1 L baffled DeLong Erlenmeyer flasks were filled with 0.4 L ofmedia consisting of 45 g/L pea protein concentrate (labeled as 80%protein), 45 g/L rice protein concentrate (labeled as 80% protein), 1g/L carrot powder, 1 g/L malt extract, 1.8 g/L diammonium phosphate and0.7 g/L magnesium sulfate heptahydrate and sterilized in an autoclavebeing held at 120-121° C. for 90 minutes. The flasks were then carefullyplaced into a laminar flowhood and cooled for 18 hours. Each flask wasinoculated with 240 mL of culture as prepared Example 2 except thestrains used were G. lucidum, C. sinensis, I. obliquus and H. erinaceus,with two flasks per species. The flasks were shaken at 26° C. at 120 RPMwith a 1″ swing radius for 8 days, at which point they were pasteurizedas according to the parameters discussed in Example 5, desiccated,pestled and tasted. The G. lucidum product contained a typical ‘reishi’aroma, which most of the tasters found pleasant. The other samples weredeemed pleasant as well but had more typical mushroom aromas.

As compared to the control, the pasteurized, dried and powdered mediumnot subjected to sterilization or myceliation, the resulting myceliatedfood products was thought to be much less bitter and to have had a moremild, less beany aroma that was more cereal in character than beany by 5tasters. The sterilized but not myceliated product was thought to haveless bitterness than the nonsterilized control but still had a strongbeany aroma. The preference was for the myceliated food product.

Example 7

Fermentation Operation in 4,000 L Bioreactor Using Continuous Sterilizer

A 4,000 L bioreactor was filled with 2,500 L of a sterilized mediumsimilar to Example 4, consisting of 45 g/L pea protein concentrate(labeled as 80% protein), 45 g/L rice protein concentrate (labeled as80% protein), 3.6 g/l maltodextrin, 1.8 g/L carrot powder, 1.8 g/Ldiammonium phosphate, 0.7 g/L magnesium sulfate heptahydrate, 1.5 g/Lanti-foam and 0.6 g/L citric acid. Seed reactor was also prepared in 200L bioreactor with medium volume of 100 L with the following mediumcomponents: pea protein 5 g/l, rice protein 5 g/l, maltodextrin 3.0 g/l,carrot powder 1 g/l, malt extract 3 g/l and 1.25 g/l of anti-foam. Themedium was inoculated with flask process developed the same way as shownin Example 2. Inoculum was harvested when pH was 4.7+/−0.1. The 200 Lbioreactor was harvested 55 hours post-inoculation. The flasks wereharvested 11 days post-inoculation. The organism was Lentinula edodessourced from the Penn State mushroom culture collection.

Once the main fermenter was cooled it was inoculated with the 100 Linoculum from the 200 L fermentor. Fermenter had an air supply of 100 to400 L/min (0.1-0.2 VVM) and held at 26° C. The culture in the 4,000 Lvessel was harvested at 48 hours post-inoculation upon successfulvisible (mycelial pellets) and microscope checks. No pH change wasobserved during the fermentation. Material was pasteurized in thebioreactor at 65 C for 60 minutes. Fermenter was then cooled down andmaterial was harvested in sanitized 55 gallon drums and sent to spraydrying facility.

Example 8

Fermentation Operation in 10,000 L Fermenter

A 10,000-L bioreactor was prepared with the following medium componentsfor a working volume of 6,200 L. pea protein 45 g/l, rice protein 45g/l, maltodextrin 3.6 g/l, carrot powder 1.8 g/l, magnesium sulfate 0.72g/l, di ammonium phosphate 1.8 g/l, citric acid 0.6 g/l, and 1.25 g/l ofanti-foam added at the end of the charge. Medium was sterilized for 2hours at 126° C. Medium was inoculated from 2000 L fermenter with avolume of 300-350 L. The aeration was maintained between 0.13 vvm and0.25 vvm. Agitation was maintained to get a tip speed of 0.88 m/sec.Additional anti-foam of 0.25 g/l was added to contain the foaming. pH ofthe medium remained at 6.1 throughout the fermentation. Temperature forthe fermentation as maintained at 26° C. Pressure in the fermenter wasincreased from 0.1 bar to 1.2 bar during the course of fermentation tominimize the foaming. Fermentation was completed in 45-50 hours. Aftercompletion of fermentation the fermented broth was pasteurized andconcentrated to 20% and then spray dried.

The seed inoculum for the fermentation was prepared in a 2000 Lfermentor with a working volume of 530-540 L with the following medium:pea protein 5 g/l, rice protein 5 g/l, maltodextrin 3.0 g/l, carrotpowder 1 g/l, malt extract 3 g/l and 1.5 g/l of anti-foam. FermentationpH was at 5.7 at the beginning of the fermentation. Fermentation wasperformed for 60 to 70 hours when pH reached between 4.6 and 4.9. Thetip speed in the fermenter was maintained at 0.5-0.6 m/s. Aeration wasdone at 0.65-0.75 vvm. Fermenter was maintained at a pressure of 0.4-0.6bar. Seed 1 for the inoculation of fermenter 2 was prepared in 150 Lwith a working volume of 55-65 L with the following medium: pea protein5 g/l, rice protein 5 g/l, maltodextrin 3.0 g/l, carrot powder 1 g/l,malt extract 3 g/l, mango puree 3 g/l and 1.5 g/l of anti-foam.Fermentation pH was at 5.7 at the beginning of the fermentation. The tipspeed in the fermenter was maintained at 0.69 m/s and pressure wasmaintained at 0.5 bar. Aeration was done at a rate of 0.75 vvm. Theinitial pH for the fermentation was at 5.7. Fermentation was completedbetween 45 and 55 hours. Inoculum for Seed 1 was prepared with the 5flask prepared in 3 L flask with the following medium: Pea Protein 5g/l, Rice Protein 5 g/l, Maltodextrin 3.0 g/l, Carrot Powder 1 g/l, maltextract 3 g/l, mango puree 3 g/l and 1.25 g/l of anti-foam. Flask wereinoculated with 4 cm² agar and incubated between 11 and 13 days. pH ofthe flask was obtained at 4+/−2.

Example 9

Fermentation Operation in 180, 000 L Fermenter

The medium for 180,000 L bioreactor was prepared as a volume of 120,000L with the following components: pea protein 45 g/l (labeled as 80%protein), rice protein 45 g/l (labeled as 80% protein), maltodextrin 3.6g/l, carrot powder 1.8 g/l, magnesium sulfate 0.72 g/l, di ammoniumphosphate 1.8 g/l, citric acid 0.6 g/l, and 1.25 g/l of anti-foam addedat the end of the charge. The 180,000 L bioreactor was harvested at 48hours.

The inoculum for the 180,000 L bioreactor was 6,200 L from a 10,000 Lbioreactor prepared similar to the medium of Example 3. The 6,200 Lbioreactor in turn was inoculated with 65 L of culture in a 150 Lbioreactor prepared similar to the 6,200 L medium and was cultured tojust before stationary phase. The 65 L medium was inoculated with flasksof Lentinula edodes in medium similar to that of the medium of Example 3and cultured to stationary phase. These flasks had been inoculated withLentinula edodes from the Penn State mushroom culture collection andculture to stationary phase.

Example 10

Sensory Data

Eight protein powders were tested: (a) raw material (3.2 pea); (b) rawmaterial (pea); (c) raw material (rice); (d) raw material (rice); (e)myceliated material 3; (f) myceliated material 4; (g) myceliatedmaterial 4.2; and (h) myceliated material 3.2. Each protein powder wastested at 7% in water. Trained descriptive panelists used a consensusdescriptive analysis technique to develop the language, ballot and rateprofiles of the protein powders. The aroma language was as follows:

Overall aroma: the intensity of the total combined aroma; pea aroma, thearoma of dried peas/pea starch (reference; ground dried peas); beanyaroma, the aroma of beans/bean starch (reference; ground dried lentils);rice aroma, the aroma of white rice (reference, cooked minute rice);mushroom aroma, the aroma of mushrooms (reference, dried shiitakemushrooms); overripe vegetable aroma, the aroma of soft overripevegetables; and cardboard aroma, the aroma of pressed wet cardboard(reference: wet pressed cardboard).

The taste language was as follows: sweet, taste on the tongue stimulatedby sugar in solution (reference, Domino Sugar in distilled water); sour,acidic taste on the tongue associated with acids in solution (reference,citric acid in distilled water); umami, the savory taste of MSG(reference; MSG in distilled water); bitter, basic taste on tongueassociated with caffeine solutions (reference, caffeine powder indistilled water); astringent, the drying, puckering feeling associatedwith tannins (reference Mott's Apple Juice (40) Welch's Grape Juice(75)).

Flavor language was as follows: overall flavor, the composite intensityof all flavors as experienced while drinking the product; overripevegetable, the flavor of soft overripe vegetables; pea, the flavor ofdried peas/pea starch (reference: ground dried peas); beany, the flavorof beans/bean starch (reference: ground dried lentils; canned garbanzobeans); rice, the flavor of white rice (reference: cooked minute rice);mushroom, the flavor of mushrooms (reference: dried shiitake mushrooms);soapy, reminiscent of soap; chalky, the flavor associated with chalk andcalcium (reference: citrucel gummies); cardboard, the flavor of pressedwet cardboard (reference: wet pressed cardboard); earthy, the flavor offresh earth/dirt (reference: potting soil).

The raw pea product prior to myceliation has a pea aroma with no rice ormushroom aroma. The rice samples prior to myceliation have rice aromawith no pea or mushroom aroma. After myceliation, these samples havemushroom aroma and no pea or rice aroma, respectively. There is alsoincreased umami flavor in the myceliated samples.

Example 11

Eight (8) 1 L baffled DeLong Erlenmeyer flasks were filled with 0.500 Lof the following 8 different media, after the manner of Example 1, seeTable 2:

TABLE 2 Component Medium 1 Medium 2 Medium 3 Medium 4 Medium 5 Medium 6Medium 7 Medium 8 Pea protein 1 (g/L) 54 54 49.5 54 54 54 0 54 Chickpeapowder (g/L) 36 36 22.5 36 36 36 36 36 Rice protein (g/L) 0 0 18 0 0 0 00 Magnesium sulfate (g/L) 0.72 0.72 0.72 0.72 0.72 0.72 0.72 0.72Diammonium phosphate (g/L) 1.8 1.8 1.8 1.8 1.8 1.8 1.8 1.8 Citric acid(g/L) 1.5 1.5 1.5 1.5 0.6 0.9 1.5 1.5 Carrot powder (g/L) 1.8 1.8 1.81.8 1.8 1.8 0 1.8 Anti-foam 1 (g/L) 1.25 0 1.25 1.25 1.25 1.25 1.25 1.25(organic polymer based) Pea protein 2 (g/L) 0 0.1 0 0 0 0 54 0 Anti-foam2 (g/L) 0 0.1 0 0 0 0 0 0 (silicone based) Vegetable juice (mL/L) 0 0 00 0 0 5 0

The flasks were covered with a stainless-steel cap and steam sterilized.The flasks were carefully transferred to a clean HEPA laminar flow hoodwhere they cooled for 4 hours and each were inoculated with 5% of 10-dayold submerged Lentinula edodes. All 8 flasks were placed on a shakertable at 150 rpm with a swing radius of 1″ at room temperature andallowed to incubate for 3 days. Plating aliquots of each sample on LBand petri film showed no contamination in any flask. The pH changesduring processing is shown below, and is essentially the same (withinthe margin of error of the pH meter). See Table 3.

TABLE 3 Medium pH, T = 0 pH T = 3 days 1 6.09 6.04 2 6.00 5.92 3 5.905.83 4 6.01 5.97 5 6.56 6.35 6 6.38 6.23 7 5.79 5.79 8 6.05 5.93

Top performing recipes in sensory from these 8 media were media 5 and 7.Bitterness and sourness were evaluated and these two media showed thebest results, although all media exhibited reduced undesirable flavorsand reduced aromas, such as reduced beany aroma, pea aroma, or ricearoma and reduced beany taste, pea taste, rice taste, and bitter taste.The sensory evaluation included 15 tasters, all tasting double-blind,randomized samples and providing a descriptive analysis. These recipeswere further evaluated for strain screening work as described in Example12.

Example 12

Eight (8) 1 L baffled DeLong Erlenmeyer flasks were filled with 0.500 Lof medium consisting of the 2 best medium as described in example 11 (4flasks for each medium). These two media were inoculated with fourdifferent species: Lentinula edodes, Boletus edulis, Pleurotussalmoneostramineus and Morchella esculenta. See Table 4.

TABLE 4 Component Medium 1 Medium 2 Pea protein 1 (g/L) 54 0 Chickpeapowder (g/L) 36 36 Magnesium sulfate (g/L) 0.72 0.72 Diammoniumphosphate (g/L) 1.8 1.8 Citric Acid (g/L) 0.6 1.5 Carrot powder (g/L)1.8 1.8 Pea protein 2 (g/L) 0 54 Anti-foam 2 (g/L) 0.1 0.1

The flasks were covered with a stainless-steel cap and sterilized in anautoclave. The flasks were carefully transferred to a clean HEPA laminarflow hood where they cooled for 4 hours and inoculated with 5% of 10-dayold submerged aliquots of each species. All 8 flasks were placed on ashaker table at 150 rpm with a swing radius of 1″ at room temperatureand incubated for 3 days at which point pH was measured and issummarized below (see Table 5):

TABLE 5 Media Species pH = To pH = 3 days 1 Lentinula edodes 6.55 6.38 1Boletus edulis 6.58 6.45 1 Pleurotus salmoneostramineus 6.55 6.44 1Morchella esculenta 6.55 5.42 2 Lentinula edodes 5.77 5.71 2 Boletusedulis 5.77 5.74 2 Pleurotus salmoneostramineus 5.76 5.88 2 Morchellaesculenta 5.77 5.36

Plating aliquots of each sample on petri film showed no contamination inany flask. Bitterness and sourness were evaluated and these two mediashowed the best results, although all media exhibited such as reducedbeany aroma, pea aroma, or rice aroma and reduced beany taste, peataste, rice taste, and bitter taste. The results that were obtainedshowed that Boletus edulis performed better than other species for lowersourness and bitterness. M esculenta did not perform well.

Example 13

A 7 L bioreactor was filled with 4.5 L of a medium consisting of themedium as described in following table (see Table 6):

TABLE 6 Component Medium 1 Medium 2 Medium 3 Pea protein 1 (g/L) 45 4558.5 Rice Protein (g/L) 45 45 31.5 Anti-foam 2 (g/L) 1.25 1.25 1.25

In this experiment, excipients other than an anti-foam were omitted fromthe fermentation medium, and only rice protein, pea protein, andanti-foam were used as the medium. In previous examples, excipients suchas magnesium sulfate, diammonium phosphate (which functions at least inpart as a buffer), citric acid, carrot powder, were used and are omittedhere. It was theorized that omission of these excipients will encouragethe culture to convert protein metabolically and not proliferate. Openports on the bioreactor were wrapped in foil and the vessel wassubsequently sterilized in an autoclave. The bioreactors were carefullytransferred to a clean bench in a cleanroom, setup and cooled for 4-6hours. The bioreactor was inoculated with 5%, 10% and 7.5% of inoculantof L. edodes from a 12-day old flask. Fermentation for these batches wascompleted in 44 hours, 24 hours and 30 hours respectively for medium 1,medium 2 and medium 3. A microscope check was done to ensure thepresence of mycelium (mycelial pellets were visible by the naked eye)and the culture was plated on LB media to ascertain the extent of anybacterial contamination and none was observed. See FIGS. 4, 5, and 6.These cultures were pasteurized for 60 minutes at 65° C. andorganoleptic taste assessments were conducted. Following tablesummarizes the pH at the harvest (see Table 7):

TABLE 7 Component Medium 1 Medium 2 Medium 3 pH t = 0 6.8 6.83 6.8 pHHarvest 6.56 6.68 6.58 Delta pH 0.24 0.15 0.22 Harvest time (hours) 2430 44

Microscopic examination of these different inoculum and protein sampleswas done and it suggested growth even for medium 1 at 24 hoursfermentation. Another interesting finding for this study was a modest pHchange of up to 0.25 units. This could be explained by the fact that themedium omitted the buffering compound diammonium phosphate from themedium

Bitterness and sourness were evaluated and these two media showed thebest results, although all media exhibited reduced beany aroma, peaaroma, or rice aroma and reduced beany taste, pea taste, rice taste, andbitter taste.

STATEMENTS REGARDING INCORPORATION BY REFERENCE AND VARIATIONS

All references throughout this application, for example patent documentsincluding issued or granted patents or equivalents; patent applicationpublications; and non-patent literature documents or other sourcematerial; are hereby incorporated by reference herein in theirentireties, as though individually incorporated by reference, to theextent each reference is at least partially not inconsistent with thedisclosure in this application (for example, a reference that ispartially inconsistent is incorporated by reference except for thepartially inconsistent portion of the reference).

The terms and expressions which have been employed herein are used asterms of description and not of limitation, and there is no intention inthe use of such terms and expressions of excluding any equivalents ofthe features shown and described or portions thereof, but it isrecognized that various modifications are possible within the scope ofthe invention claimed. Thus, it should be understood that although thepresent invention has been specifically disclosed by preferredembodiments, exemplary embodiments and optional features, modificationand variation of the concepts herein disclosed may be resorted to bythose skilled in the art, and that such modifications and variations areconsidered to be within the scope of this invention as defined by theappended claims. The specific embodiments provided herein are examplesof useful embodiments of the present invention and it will be apparentto one skilled in the art that the present invention may be carried outusing a large number of variations of the devices, device components,methods steps set forth in the present description. As will be obviousto one of skill in the art, methods and devices useful for the presentmethods can include a large number of optional composition andprocessing elements and steps.

Whenever a range is given in the specification, for example, atemperature range, a time range, or a composition or concentrationrange, all intermediate ranges and subranges, as well as all individualvalues included in the ranges given are intended to be included in thedisclosure. It will be understood that any subranges or individualvalues in a range or subrange that are included in the descriptionherein can be excluded from the claims herein.

All patents and publications mentioned in the specification areindicative of the levels of skill of those skilled in the art to whichthe invention pertains. References cited herein are incorporated byreference herein in their entirety to indicate the state of the art asof their publication or filing date and it is intended that thisinformation can be employed herein, if needed, to exclude specificembodiments that are in the prior art. For example, when composition ofmatter are claimed, it should be understood that compounds known andavailable in the art prior to Applicant's invention, including compoundsfor which an enabling disclosure is provided in the references citedherein, are not intended to be included in the composition of matterclaims herein.

As used herein, “comprising” is synonymous with “including,”“containing,” or “characterized by,” and is inclusive or open-ended anddoes not exclude additional, unrecited elements or method steps. As usedherein, “consisting of” excludes any element, step, or ingredient notspecified in the claim element. As used herein, “consisting essentiallyof” does not exclude materials or steps that do not materially affectthe basic and novel characteristics of the claim. In each instanceherein any of the terms “comprising”, “consisting essentially of” and“consisting of” may be replaced with either of the other two terms. Theinvention illustratively described herein suitably may be practiced inthe absence of any element or elements, limitation or limitations whichis not specifically disclosed herein.

One of ordinary skill in the art will appreciate that startingmaterials, biological materials, reagents, synthetic methods,purification methods, analytical methods, assay methods, and biologicalmethods other than those specifically exemplified can be employed in thepractice of the invention without resort to undue experimentation. Allart-known functional equivalents, of any such materials and methods areintended to be included in this invention. The terms and expressionswhich have been employed are used as terms of description and not oflimitation, and there is no intention that in the use of such terms andexpressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention claimed.Thus, it should be understood that although the present invention hasbeen specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the appended claims.

We claim:
 1. A method to prepare a myceliated high-protein food product,comprising the steps of: providing an aqueous medium comprising ahigh-protein material, wherein the aqueous medium comprises at least 50%(w/w) protein on a dry weight basis, wherein the media comprises atleast 50 g/L protein and wherein the high protein material is from aplant source; inoculating the medium with a fungal culture, wherein thefungal culture comprises Lentinula edodes, Agaricus spp., Pleurotusspp., Boletus spp., or Laetiporus spp., and culturing the medium toproduce a myceliated high-protein food product; wherein the myceliatedhigh-protein food product has reduced undesirable flavors and reducedundesirable aromas compared to the high-protein material that is notmyceliated.
 2. The method of claim 1, wherein the Laetiporus spp. isLaetiporus sulfureus.
 3. The method of claim 1, wherein the Pleurotusspp. comprises Pleurotus ostreatus, Pleurotus salmoneostramineus(Pleurotus djamor), Pleurotus eryngii, or Pleurotus citrinopileatus. 4.The method of claim 1, wherein the Pleurotus spp. comprises Pleurotusostreatus or Pleurotus salmoneostramineus (Pleurotus djamor).
 5. Themethod of claim 1, wherein the Boletus spp. comprises Boletus edulis andAgaricus spp. comprises Agaricus blazeii, Agaricus bisporus. Agaricuscampestris, Agaricus subrufescens, Agaricus brasiliensis or Agaricussilvaticus.
 6. The method of claim 1, wherein the fungal culture is asubmerged fungal culture.
 7. The method of claim 1, wherein thehigh-protein material is at least 70% (w/w) protein on a dry weightbasis.
 8. The method of claim 1, wherein the aqueous media comprisesbetween 50 g/L protein and 200 g/L protein.
 9. The method of claim 1,wherein the high-protein material is a protein concentrate or a proteinisolate.
 10. The method of claim 9, wherein the high-protein material isfrom a plant source.
 11. The method of claim 10, wherein the plantsource comprises pea, rice, or combinations thereof.
 12. The method ofclaim 10, wherein the plant source comprises chickpea.
 13. The method ofclaim 10, wherein the plant source comprises pea.
 14. The method ofclaim 10, wherein the plant source comprises rice.
 15. The method ofclaim 1, wherein the myceliated high-protein food product is sterilizedor pasteurized prior to the inoculating step.
 16. The method of claim 1,wherein the method further comprises the step of drying the myceliatedhigh-protein food product.
 17. The method of claim 1, wherein themyceliated high-protein food product has enhanced desirable flavors andenhanced desirable aromas.
 18. The method of claim 1, wherein theaqueous medium comprises, on a dry basis, dry carrot powder, dry maltextract, diammonium phosphate, magnesium sulfate, and citric acid. 19.The method of claim 1, wherein the aqueous media consists of, on a dryweight basis, a protein source selected from the group consisting of arice protein source, a pea protein source, and a chickpea proteinsource, and an anti-foam component.
 20. The method of claim 19, whereinthe anti-foam component is a food-grade silicone oil emulsion componentor an organic polymer-based anti-foam component.
 21. The method of claim19, wherein the pH of the fungal culture has a change of less than 0.5pH units during the myceliation step.
 22. The method of claim 21,wherein the pH of the fungal culture has a change of less than 0.3 pHunits during the myceliation step.
 23. The method of claim 1, whereinthe culturing step is carried out until the dissolved oxygen in themedia reaches between 80% and 90% of the starting dissolved oxygen. 24.The method of claim 1, wherein the pH of the fungal culture does notchange during processing.
 25. The method of claim 1, wherein the pH ofthe fungal culture has a change of less than 0.3 pH units during themyceliation step.
 26. The method of claim 1, wherein the pH of thefungal cultures has a change of less than 0.2 pH units during themyceliation step.
 27. The method of claim 1, wherein the reducedundesirable flavor is a pea flavor or a bitterness flavor.
 28. Themethod of claim 1, wherein the reduced undesirable aroma is a beanyaroma or a rice aroma.
 29. A myceliated food product made by the methodof claim
 1. 30. A composition comprising a myceliated high-protein foodproduct, wherein the myceliated high-protein food product is at least50% (w/w) protein on a dry weight basis, wherein the myceliated highprotein food product is derived from a plant source, wherein themyceliated high protein product is myceliated by a fungal culturecomprising Lentinula edodes, Agaricus blazeii, Pleurotus spp., Boletusspp., or Laetiporus spp. in a media comprising at least 50 g/L protein,and wherein the myceliated high protein food product has reducedundesirable flavor and reduced undesirable aroma compared with anon-myceliated food product.
 31. The composition of claim 30, whereinthe myceliated high-protein food product is at least 70% (w/w) proteinon a dry weight basis.
 32. The composition of claim 30, wherein theplant source is pea, rice, or combinations thereof.
 33. The compositionof claim 30, wherein the myceliated high-protein food product is in theform of a powder.
 34. The composition of claim 30, wherein themyceliated high-protein food product is produced according to the methodof claim
 1. 35. The composition of claim 30, wherein the myceliatedhigh-protein food product has enhanced desirable flavors and enhanceddesirable aromas.